The thyroid gland is an endorine gland situated at the base of the neck which synthesizes two peptidic hormones, T.sub.3 triiodothyronine and T.sub.4 thyroxine, by the condensation of two molecules of iodized thyrosine, namely monoiodothyrosine and diiodothyrosine.
The synthesis takes place from a thyroid protein, thyroglobulin, which is also the reserve form of the thyroid hormones. These hormones are released into the circulation by a proteolytic process, the hormones being conveyed by three seric proteins: TBG (thyroxine-bonding globulin), TBPA (thyroxine-bonding prealbumin) and albumin. However, the free and bonded forms of the hormones exist in the blood in a state of dynamic equilibrium governed by the law of mass action.
The thyroid hormones exert their activity on the majority of tissues, except the brain. For example, they stimulate the oxygen consumption of the majority of cells, take part in regulating the metabolism of the lipids and glucids and are necessary for the development of normal growth. However, it is generally assumed that only the free forms of the hormone are biologically active and can enter the cells to exert their physiological action.
Processes allowing the quantitative determination not only of the total concentration of the T.sub.3 and T.sub.4 hormones but also of the concentration of these hormones in free form are very advantageous for diagnosing hyperthyroidism or hypothyroidism, since the determination of the total T.sub.4 and/or T.sub.3 rate may lead to errors in the diagnosis of hyperthyroidism or hypothyroidism caused by an alteration in the transporting proteins. The fact is that the concentration of bonding proteins may increase, for example, during pregnancy, liver diseases and when oestrogens or opiates are taken, in which case the total T.sub.4 or T.sub.3 rate increase, at the same time, and this may lead to a diagnosis of hyperthyroidism, although the rates of free T.sub.3 or T.sub.4 are normal and so is the patient's thyroid gland.
Conversely, the total T.sub.3 or T.sub.4 rate can be reduced by a reduction in the rate of the bonding proteins, and this might lead to a diagnosis of hypothyroidism, although the rate of free T.sub.3 or T.sub.4 is normal.
It is therefore preferably to be able to determine the rates of free T.sub.3 or T.sub.4 to better appreciate the condition of the patient, since such rates can be determined either directly or from the total T.sub.3 and T.sub.4 concentrations and the index of saturation of the carrier proteins.
Low rates of the T.sub.3 and T.sub.4 thyroid hormones found in human or animal sera require the use of sensitive methods of quantitative determination.
The methods currently used for quantitative determinations of this kind adopt immunological techniques, including, first of all, radio-immunological quantitative determinations using T.sub.4 or T.sub.3 labelled with iodine.sup.125. More recently, techniques have been developed for enzyme-immunological quantitative determination, in either the heterogeneous or homogeneous phase.
The methods in the homogeneous phase include quantitative determinations based on the polarization of fluorescence, the transfer of fluorescence or an inhibition of the enzyme. These determinations have the advantage that they can be completely automated, but they require the use of sophisticated equipment.
The methods in the heterogeneous phase include a process for the determination of the free or total T.sub.3 or T.sub.1 which consists in bringing into competition T.sub.3 or T.sub.4 and a known quantity of T.sub.3 or T.sub.4 labelled with an enzyme for a limited number of specific antibody sites of the hormone (H. V. Weetall et al., Clin. Chem., Vol. 28, no. 4 (1982), pp. 666-671; SCHALL et al, Clin. Chem., 1978, 24, 1801-1804; and ALBERT et al., Ed. S. B. Pal, 1978, pp. 153-174). To perform the same kind of determination in the heterogeneous phase, the hormone to be determined and a predetermined quantity of hormone fixed on a solid support might be put into competition for a limited quantity of active sites of a labelled antibody specific to the hormone. (Masao Ito et al., Clin. Chem., 30/10, 1682-1685 (1984) and Gnemmi et al., Enzyme Labelled Immunoassay of Hormones and Drugs, 1978, Walter de Gruyter and Co., Berlin-New-York).
However, these two methods of quantitative determination in the heterogeneous phase are difficult to implement with the T.sub.3 and T.sub.4 hormones, which generally lose their antigen activity when they are fixed on a solid support, or their biological and/or antigen activity when they are coupled with a macromolecule. Moreover, when this method is to be used for the quantitative determination of T.sub.3 and/or T.sub.4 in the free form, the labelled T.sub.3 and/or T.sub.4 molecules must not be recognized by the proteins transporting the T.sub.3 and T.sub.4 hormones.
On the other hand, the determination might be made by coupling the T.sub.3 or T.sub.4 hormone with a powder, but in that case, the performance of the determination raises problems of washing.